Globins and hypoxia adaptation in the goldfish, Carassius auratus Anja Roesner 1 , Stephanie A. Mitz 1,2 , Thomas Hankeln 3 and Thorsten Burmester 2 1 Institute of Zoology, University of Mainz, Germany 2 Institute of Zoology, University of Hamburg, Germany 3 Institute of Molecular Genetics, University of Mainz, Germany Many freshwater environments are characterized by spatial, temporal or seasonal fluctuations in oxygen availability. Therefore, various fish species have evolved physiological, anatomical and behavioral mechanisms for coping with extended periods of hypoxia [1–8]. These strategies include O 2 saving by reduction of the metabolic rate, improved O 2 uptake by enhanced ventilation, aquatic surface respiration, expansion of the gill surface and the increased O 2 affinity of hemoglobin [9,10]. Cyprinid fishes of the genus Carassius (the crucian carp Carassius carassius and its domestic Asian form, the goldfish C. auratus) routinely experience hypoxia and even anoxic phases in their environment of isolated ponds. Carassius dis- plays remarkable tolerance against O 2 deprivation. This tolerance is conveyed by high glycogen stores in brain and liver, increased buffering capacities, meta- bolic rate depression and the ability to convert the lactate produced by anaerobic glycolysis into ethanol, which is excreted via the gills [2,3,8,11–14]. In recent years, hypoxia has received much attention in biomedical research [8,15] and, because of global Keywords cytoglobin; goldfish; hemoglobin; myoglobin; neuroglobin Correspondence T. Burmester, Institute of Zoology, University of Hamburg, Biozentrum Grindel, Martin-Luther-King-Platz 3, D-20146 Hamburg, Germany Fax: +49 40 42838 3937 Tel: +49 40 42838 3913 E-mail: thorsten.burmester@uni-hamburg.de Database The nucleotide sequences have been sub- mitted to the GenBank database ⁄ EMBL Data Bank under accession numbers AM933143 (Hba), AM933144 (Hbb), AM747267 (Mb1), AM747268 (Mb2), AM933145 (Ngb) and AM933146 (Cygb1) (Received 6 March 2008, revised 6 May 2008, accepted 15 May 2008) doi:10.1111/j.1742-4658.2008.06508.x Goldfish (Carassius auratus) may survive in aquatic environments with low oxygen partial pressures. We investigated the contribution of respiratory proteins to hypoxia tolerance in C. auratus. We determined the complete coding sequence of hemoglobin a and b and myoglobin, as well as partial cDNAs from neuroglobin and cytoglobin. Like the common carp (Cypri- nus carpio), C. auratus possesses two paralogous myoglobin genes that duplicated within the cyprinid lineage. Myoglobin is also expressed in non- muscle tissues. By means of quantitative real-time RT-PCR, we determined the changes in mRNA levels of hemoglobin, myoglobin, neuroglobin and cytoglobin in goldfish exposed to prolonged hypoxia (48 h at Po 2  6.7 kPa, 8 h at Po 2  1.7 kPa, 16 h at Po 2  6.7 kPa) at 20 °C. We observed small variations in the mRNA levels of hemoglobin, neuroglobin and cytoglobin, as well as putative hypoxia-responsive genes like lactate dehydrogenase or superoxide dismutase. Hypoxia significantly enhanced only the expression of myoglobin. However, we observed about fivefold higher neuroglobin protein levels in goldfish brain compared with zebrafish, although there was no significant difference in intrinsic myoglobin levels. These observations suggest that both myoglobin and neuroglobin may con- tribute to the tolerance of goldfish to low oxygen levels, but may reflect divergent adaptive strategies of hypoxia preadaptation (neuroglobin) and hypoxia response (myoglobin). Abbreviations ARP, acidic ribosomal phosphoprotein P0; Cygb, cytoglobin; GbX, globin X; Hb, hemoglobin; LDH-A, lactate dehydrogenase A; Mb, myoglobin; Ngb, neuroglobin; SOD, superoxide dismutase. FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS 3633 warming, has become an important environmental concern [16,17]. Fish have become a prime model to investigate hypoxia tolerance strategies at the organism level. Alternative metabolic pathways, as well as other physiological responses are associated with major changes in gene expression patterns. For example, genes encoding enzymes of the glycolytic pathway and fermentation are expressed more strongly after long- term hypoxia in some fish species [9,18,19]. By con- trast, genes required for oxidative energy production in the tricarboxylic acid cycle or the respiratory chain, or for the highly energy-consuming translation process were found to be repressed. Hypoxia may even cause developmental arrest, which is reflected by the repres- sion of growth or cell cycle-associated genes [19,20]. Hypoxia also affects the O 2 -binding respiratory proteins, which are represented in vertebrates by the globin superfamily [21–23]. Globins are small globular proteins that bind to O 2 by virtue of a heme-bound Fe 2+ ion. To date, five types of globins have been identified in fish. Hemoglobin (Hb) is included in the red blood cells and serves for the transport of O 2 within the circulatory system. Hb is a heterotetramer that consists of two a- and two b-chains. Monomeric myoglobin (Mb) supplies O 2 within the striated muscle and heart of most vertebrates [24]. Whereas Mb is a single copy gene in most species, the common carp (Cyprinus carpio) possesses two paralogous Mb genes (Mb1 and Mb2) [25]. Surprisingly, Mb1 is ubiquitously expressed in various tissues, whereas Mb2 is restricted to the brain. Neuroglobin (Ngb) is located in the cen- tral and peripheral nervous systems [26], the retina [27] and some endocrine tissues [28]. The exact role of Ngb remains uncertain; it may supply O 2 to metabolically active neurons, although other functions such as the detoxification of noxious reactive oxygen or nitrogen species are conceivable [29–31]. Cytoglobin (Cygb) is located in the fibroblast cell lineage as well as in dis- tinct populations of neurons [32,33]. The function of Cygb may be related to reactive oxygen species detoxi- fication or the supply of O 2 to particular enzymatic reactions [30,33]. Fish possess two paralogous Cygb genes, which show divergent expression in neurons and non-neuronal tissues [34]. The most recently identified vertebrate globin, which has been referred to as glo- bin X (GbX), is restricted to fish and amphibians [35,36]. The physiological role of GbX, which is expressed at low levels in a broad range of tissues, is currently unknown. Because hypoxia reduces the availability of O 2 to mitochondria and respiratory proteins, it can be assumed that low O 2 levels change the expression of globins. Previously, we investigated the effect of hypoxia on globin mRNA and protein levels in the zebrafish Danio rerio [23]. Zebrafish exhibit moderate tolerance to hypoxia, surviving extended periods at Po 2  4 kPa. It might be expected that increased expression of respiratory proteins should be advanta- geous at low O 2 partial pressures, however, we found different globin responses. Whereas Hb mRNA levels decreased under hypoxia, Mb and Ngb protein and mRNA levels increased significantly. The data suggest that these globins are involved in conveying hypoxia tolerance to zebrafish. Here we investigate the response of globin levels in the extremely hypoxia-tolerant goldfish. Results Cloning and analyses of goldfish globins Partial and complete cDNA sequences of goldfish Hba,Hbb, Mb1, Mb2, Ngb and Cygb1 were obtained by RT-PCR from RNA extracted from various tissues (Fig. 1 ). The complete coding sequences, including the 5¢- and 3¢-ends, of Hba,Hbb and Mb1 were then obtained from a mixed tissue cDNA library (supple- mentary Figs S1–S3). The cDNA of Mb2 was obtained by RT-PCR (supplementary Fig. S4). The 3¢-end of the coding sequence of Ngb was missing, and we obtained only the middle part of Cygb1 (supplemen- tary Figs S5 and S6). Cygb2 could not be obtained by RT-PCR. Because our study was mainly aimed at investigating the regulation of globin expression, which requires only fragments of globin cDNA, we ignored the missing parts of the coding sequences. The coding sequence of GbX had been obtained in a previous study [36]. We first compared goldfish globin sequences with their zebrafish orthologs (Fig. 1). Both Hb cDNAs represent adult chains, whereas embryonic Hbs were not considered in this study. The Hba chain we obtained by screening the cDNA library is 97% identi- cal at the nucleotide level to the goldfish Hba cDNA sequence available in the databases (accession number AF528157), suggesting allelic variation or the presence of multiple isoforms in the C. auratus genome. Gold- fish and zebrafish Hba proteins are 87.4% identical (99.3% similar; considering isofunctional replace- ments). The Hbb chains of these two fish species are 92.6% identical ⁄ 98.0% similar; within the overlapping regions, the Cygb1 proteins of the two species are 79.7% identical ⁄ 95.8% similar and Ngb is 92.4% iden- tical ⁄ 98.1% similar. GbX proteins are highly con- served, with scores of 98.0% identity and 99.0% similarity. Goldfish globins under hypoxia A. Roesner et al. 3634 FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS Recently, Fraser et al. [25] found two distinct Mb sequences in the common carp (Cy. carpio). Although Mb1 could be readily obtained by screening the cDNA library, Mb2 was identified by RT-PCR using oligonu- cleotide primers designed according to the C. carpio Mb2 sequence. Goldfish and carp Mb1 proteins are 93.9% identical and 98.0% similar; the Mb2 proteins are 88.4% identical and 96.6% similar (Fig. 2 ). The paralogs are  78% identical and  90% similar. When compared with zebrafish Mb, the scores for goldfish Mb1 are 81.6% identical ⁄ 91.8% similar, and for Mb2 78.2% identical ⁄ 88.4% similar. Using zebra- fish anti-Mb serum, we examined the presence of Mb protein in goldfish organs (Fig. 3 ). We detected Mb protein in all investigated tissues (brain, gills, heart, liver, kidney and swimbladder). As expected, the Mb signal was strongest in heart (note the different amounts of total proteins applied per lane), but we also observed apparently high Mb concentration in the goldfish gills. Changes in gene expression in hypoxic goldfish In previous studies with zebrafish, we employed acidic ribosomal phosphoprotein P0 (ARP) as the nonregu- lated reference gene [23]. Fragments of ARP were amplified from goldfish RNA using primers that had been designed according to known zebrafish sequences. Fig. 1. Comparison of zebrafish (Dre) and goldfish (Cau) globins. The amino acid sequences from Hba (HbA), Hbb (HbB), Mb, Ngb, Cygb and GbX were aligned. The secondary structure of human neuroglobin is superimposed in the upper row, with alpha-helices designated A–H, the globin consensus numbering is given below the sequences. Strictly conserved amino acids are shaded in gray. Invariable (B12.2 and G7.0) and variable (E10.2 and H10.0) intron positions in vertebrate globin genes are indicated by arrows in the upper row. Fig. 2. Comparison of zebrafish (Dre) myoglobin and the paralogous carp (Cca) and goldfish (Cau) Mb1 and Mb2. The predicted secondary structure of zebrafish Mb is superimposed in the upper row, with alpha-helices designated A–H, the globin consensus numbering is given below the sequences. Strictly conserved amino acids are shaded in gray, functionally important residues (PheCD1, HisE7 and HisF8) are white on a black background. A. Roesner et al. Goldfish globins under hypoxia FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS 3635 ARP mRNA levels remained constant in most tissues; however, in brain and eye we found significant upregu- lation of ARP mRNA by a factor of  1.7 (P < 0.01). Therefore, all expression levels were subse- quently normalized according to total RNA content. However, none of the conclusions presented here was affected if expression levels were normalized according to ARP (data not shown). We applied mixed chronic and acute hypoxia regimes that lasted 3 days. First, mild chronic hypoxia was induced by a reduction in Po 2 to  6.7 kPa for 48 h. Acute hypoxia was achieved at Po 2  1.7 kPa for 8 h, followed by Po 2  6.7 kPa for an additional 16 h before RNA extraction. Reduction of Po 2 to close anoxia (< 0.5 kPa) led to the death of all experi- mental animals within 16 h. Normoxic controls were kept at Po 2  18.4 kPa. The expression levels of lac- tate dehydrogenase A (LDH-A), Hba,Hbb, Cygb1 and Mb1 were first analyzed in goldfish body (car- casses without heart, brain and eyes) (Fig. 4A ). Thus the majority of tissue represents skeletal muscle, but also includes blood vessels. We observed a mild ( 25%) downregulation of Hba,Hbb and Cygb1 mRNA under hypoxia, which was, however, not signif- icant. LDH-A levels were unchanged, whereas Mb1 mRNA was found to be heavily upregulated ( 18- fold; P < 0.05). In heart, Mb1 mRNA levels were essentially unaffected (Fig. 4B). In brain, we observed a twofold increase in Mb2 mRNA (P < 0.01), although Mb1, Ngb, LDH-A and superoxide dismu- tase (SOD)-1 mRNA levels remained essentially con- stant (Fig. 4C). No significant changes in mRNA levels of the investigated genes were observed in total eye (Fig. 4D). Quantitative western blotting To compare the protein levels of Ngb and Mb from goldfish and zebrafish, we performed quantitative wes- tern blotting. We used specific antibodies that had Brain Gill Heart Liver Kidney Swimbladder 18 kDa Fig. 3. Myoglobin expression in goldfish tissues. Protein extracts from selected goldfish tissues were analyzed by western blotting employing a zebrafish anti-Mb serum. Protein extracts (100 lg) were applied on each lane for brain, gills, liver and kidney; 50 lg was loaded for heart and swimbladder plus associated tissues. The position of the 18 kDa molecular mass marker is indicated on the right side. A B C D Fig. 4. Expression of goldfish globins at different oxygen levels. mRNA quantities were determined by quantitative real-time RT- PCR. The white columns represent mRNA levels from goldfish kept at normoxia (P O 2  18.4 kPa), gray columns are mRNA levels from goldfish kept at hypoxia (48 h P O 2  6.7 kPa, 8 h PO 2  1.7 kPa, 16 h at P O 2  6.7 kPa). RNA was extracted from carcasses (A), heart (B), brain (C) or eye (D). Bars represent SD. The significance of the data was estimated with a Student’s t-test, with n =4. **P < 0.01. Gene abbreviations: Cygb, cytoglobin; HbA, hemo- globin a; HbB, hemoglobin b; LDH-A, lactate dehydrogenase A; Mb1, myoglobin 1; Mb2, myoglobin 2; Ngb, neuroglobin; SOD-1, superoxide dismutase-1. Goldfish globins under hypoxia A. Roesner et al. 3636 FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS been raised against recombinant zebrafish Ngb or Mb. First, we evaluated Ngb and Mb protein levels in normoxic and hypoxic goldfish. No discernable changes of Ngb in protein extracts from brain and eye, or Mb in extracts from brain, heart and liver were observed (supplementary Fig. S7). For interspecific comparisons, total proteins were extracted from brains, total eyes and hearts of individual goldfish and zebra- fish specimens that had been kept under normoxic con- ditions. We applied a constant amount of total protein extracts to SDS ⁄ PAGE (100 lg per lane for brain and eye; 15 lg for heart) and western blotting (Fig. 5 ). To avoid variations due to different treatments, samples from both species were applied to the same gel and transferred to a single membrane, which was then incubated with the antibodies. We observed an approx- imately fivefold higher Ngb protein level in the gold- fish brain compared with zebrafish brain (Fig. 5A). The difference was highly significant, as estimated by a Student’s t-test (P < 0.001). In protein extracts from goldfish eyes, Ngb protein levels were 3.2-fold higher than in zebrafish eyes (P < 0.05). We observed no dif- ferences in Mb levels in goldfish and zebrafish, either in brain or in heart (Fig. 5B). Discussion Many fish species have evolved strategies that allow them to survive phases of acute and chronic hypoxia [2,5–8]. Carassius species are particularly hypoxia toler- ant, with various mechanisms that help them to better survive hypoxia and even anoxia [2,11,37]. We recently analyzed the expression regulation and putative roles of the various globins in another, less hypoxia-tolerant species of the Cypriniformes, the zebrafish D. rerio [23]. Comparison of these data with those obtained from C. auratus will help to delineate the contribution of globin expression regulation to hypoxia tolerance. In particular, we focused on Mb and Ngb; although a respiratory role for Mb is well documented, our results provide better understanding of the physiological role of the recently discovered Ngb. Two paralogous Mb genes in Cyprininae Mb is an intracellular respiratory protein that mainly facilitates the diffusion of O 2 from the capillaries to the mitochondria and stores O 2 [37]. Until recently, it had been commonly assumed that there is only a single Mb gene in vertebrates and that Mb expression is con- fined to muscle tissue. Mb protein is present at high concentrations in the skeletal and heart muscles of most vertebrates [37], but has also been identified in smooth muscle [38,39]. However, Fraser et al. [25] demonstrated that the common carp possesses two paralogous Mb genes, of which one (Mb1) is ubiqui- tously present also in nonmuscle tissues; Mb2 expres- sion is restricted to the carp’s brain. We confirmed these results in goldfish, which also possesses two dis- tinct Mb genes (Fig. 2) and exhibits ubiquitous expres- sion of Mb (Fig. 3). This suggests that the duplication of Mb genes and the altered expression patterns occurred before the divergence of the genera Cyprinus and Carassius (both belonging to the subfamily Cyprininae), which separated  11 million years ago [40]. As revealed by database searches, the zebrafish AB Fig. 5. Comparison of neuroglobin (A) and myoglobin (B) protein levels in goldfish and zebrafish. Protein levels were estimated by quantitative western blotting (cf. Fig. S8). Three individual zebrafish specimens and four goldfishes were used for the experi- ments. The units of the y-axis are arbitrary, with zebrafish brain = 1. Note that the rela- tive levels of Mb in brain and heart cannot be compared. ***P < 0.001; *P < 0.05 (t-test), n.s., not significant. A. Roesner et al. Goldfish globins under hypoxia FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS 3637 D. rerio genome harbors only a single Mb gene. It may be assumed that the emergence of an additional Mb gene is linked to a genome duplication event, which occurred in the Cyprininae 12–16 million years ago [40]. We observed Mb protein expression in goldfish as well as in zebrafish brain, suggesting that nonmuscle expression of Mb emerged before the diver- gence of D. rerio and Cyprininae within the lineage of the Cypriniformes. As proposed by Fraser et al. [25], the occurrence of Mb in tissues other than muscle, its hypoxia-inducible expression, as well as the occurrence of the brain-specific isoform Mb2 may be part of the strategy of the Cyprininae for better survival during prolonged periods of hypoxia. Expression-regulation of globins and their function in hypoxia As in zebrafish [23], we observed a mild downregula- tion of Hba and Hbb mRNA levels at hypoxia com- pared with normoxic controls (Fig. 4A). The hypoxia response of Hb has been investigated in various fish species, with conflicting results. For example, Timmer- man and Chapman [41] reported an increase in Hb levels in the sailfin molly (Poecilia latipinna), whereas Person Le Ruyet et al. [42] observed no difference in normoxic and hypoxic juvenile turbot (Scophthal- mus maximus) and seabream (Sparus aurata). In zebra- fish, the Hb mRNA levels decrease under hypoxia [23,41]. The contribution of the transcription regula- tion of Hb to hypoxia tolerance is species specific and may also depend on the hypoxia regime [43]. Never- theless, Carassius Hb is 50% saturated even at Po 2 = 0.33 kPa, thereby contributing to hypoxia tolerance [44]. Most likely, the O 2 -affinity of fish Hb is largely regulated on a post-translational level, i.e. via alteration of O 2 affinities by means of modulators such as ATP and GTP [45]. Therefore, alterations in Hb mRNA levels are not necessarily required. Mb1 and Mb2 were the only globin-types in goldfish to show hypoxia induction at the mRNA level. It may be assumed that enhanced expression of Mb is associated with hypoxia tolerance in goldfish. Additional Mb in various tissues increases the availability of O 2 to the respiratory chain of the mitochondria, thereby prom- oting the survival of the cells. Putative role of Ngb in preadaptation of the brain to hypoxia Altered expression levels of certain proteins may help to improve the animal’s survival under unfavorable condi- tions. For example, interspecific variations in heat shock proteins have been found in marine gastropods and have been attributed to the acclimatization to dif- ferent habitats [46,47]. The high Mb content in the mus- cles of marine mammals such as whales and seals is considered an adaptation to long-term dives [48]. Noth- ing is known about the more recently discovered Ngb. Although the localization, expression, regulation and evolution of Ngb have been thoroughly investigated in recent years, its exact role in vertebrate neurons is not well understood [29,30]. Whereas some studies point to a Mb-like role of Ngb in supplying O 2 [26,49], thereby enhancing the survival of neurons under hypoxia [50], other authors have proposed a function for Ngb in the detoxification of reactive nitrogen species and NO [51,52] or hypoxia-related signaling [53,54]. Most stu- dies agree that in vivo hypoxia does not significantly enhance Ngb expression in the mammalian brain [21]. This is not surprising, because under normal condi- tions most mammals never experience low O 2 environ- ments during their adult life. However, as already pointed out, many fish live in hypoxic environments. In fact, we previously observed an increase in Ngb levels of up to 5.7-fold in hypoxic zebrafish brain compared with normoxia controls, which suggests the involvement of this protein in hypoxia response in this fish species [23]. It is well established that the Carassius brain has evolved various strategies to survive very low oxygen levels [37]. However, C. auratus, which is actually more hypoxia-tolerant than zebrafish, does not show a hypoxia response in Ngb expression. At first sight, this is difficult to reconcile with the hypothesis that Ngb is involved in O 2 supply for respiration or has any other Po 2 -related function such as reactive oxygen species detoxification. However, we did not observe an increase in the expression of the typically hypoxia- responsive gene LDH-A, or of the reactive oxygen species-defense gene SOD, although the fact that Mb1 is heavily upregulated shows that the hypoxia regime we applied in this study actually induce changes in gene expression. As shown in Fig. 5, there is an appro- ximately fivefold higher level of Ngb protein in the goldfish brain compared with the related, less hypoxia- tolerant zebrafish. This is the first time that higher Ngb concentrations could be correlated with hypoxia tolerance, which may be interpreted as a preadaptation of the goldfish brain. A similar observation has been made in the subterranean mole rat Spalax ehrenbergi, a mammal that can survive extended periods of hypoxia without neuronal damage, and which has con- stitutively higher expression levels of Ngb compared with rats (A. Avivi, F. Gerlach, T. Burmester, E. Nevo & T. Hankeln, unpublished results). These data Goldfish globins under hypoxia A. Roesner et al. 3638 FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS provide additional support for an adaptive role of Ngb in hypoxia tolerance of neurons, and for a possible func- tion of Ngb in O 2 storage or facilitated O 2 diffusion. Distinct roles of Mb and Ngb in hypoxia adaptation Changing environmental oxygen concentrations have a significant impact on the expression of intracellular respiratory proteins, which increase the availability of O 2 to the tissues. In mammals, the results on the impact of hypoxia on Mb expression are variable [55,56]. Data on hypoxia-regulation of Ngb in mam- malian systems are also not consistent. Although Ngb was found to be more highly expressed in hypoxic cell and tissue culture systems, no changes in Ngb mRNA levels were found in whole-animal experiments [21]. Here we have demonstrated that both Mb and Ngb may contribute to the extreme hypoxia tolerance of goldfish. The increased expression of Mb under hypoxia and the high intrinsic Ngb levels in goldfish neuronal tissues agree with the proposed function of these proteins in O 2 supply. However, neuronal tissues require a consistent supply with sufficient O 2 and any shortage results in severe defects in the brains of most vertebrates. The function of the nervous system thus requires an uninterrupted O 2 supply. The high intrinsic concentration of Ngb may guarantee its immediate availability upon the onset of hypoxia, and may be part of the strategy that secures a constant flow of O 2 to the highly energy-demanding neurons. In contrast to neurons, striated muscle cells can survive via anaer- obic fermentation; therefore, a delayed increase in Mb concentration (as reflected by enhanced Mb mRNA levels) is sufficient to ensure the supply of O 2 to the muscle cells. Together with the high-affinity Hb [44], high levels of Mb and Ngb may contribute to the fact that Carassius are able to maintain normal O 2 con- sumption rates down to oxygen levels of 5–10% of air saturation in water [57]. Experimental procedures Experimental animals Adult goldfish (C. auratus L.) were purchased in a local pet shop and kept for several months in a large tank. Animals used for the hypoxia or normoxia control experiments (weighing around 4 g each) were directly transferred to a 100 L aquarium and kept at 14 h light ⁄ 10 h dark cycle and a temperature of 20 °C. Water was filtered with a thermofil- ter (Ekip 350; Hydor, Bassano del Grappa, Italy). Water quality was checked periodically (Multi Check; Amtra, Rodgau, Germany) and partial water changes were carried out when necessary. Animal handling and experiments were conducted according to a protocol that had been approved by the county government office (Bezirksregierung Rhein- hessen-Pfalz, AZ 1.5 177-07 ⁄ 021-30). Hypoxia treatment Groups of four goldfish were randomly assigned to hypoxia treatment or control groups. The animals were not fed for 24 h before or during the experiments. Hypoxia treatment was performed in a 40 L aquarium with loosely fitting covers. Water was bubbled with gas mixtures (2% O 2 in N 2 or 100% N 2 ; Air Liquide, Du ¨ sseldorf, Germany). A ther- mopump (Ekip 350; Hydor) was used to ventilate the water and to keep the temperature constant at  20 °C. O 2 par- tial pressure and temperature were measured every 15 min using an oxygen sensor (Oxi 340i, WTW, Weilheim, Germany). Hypoxia treatment was started by reducing the Po 2 to  6.7 kPa ( 50 Torr) for 48 h, Po 2  1.7 kPa ( 13 Torr) for 8 h, followed by Po 2  6.7 kPa ( 50 Torr) for additional 16 h. O 2 partial pressure remained constant (± < 0.5 kPa) during experimental time. Control animals were kept under the same conditions, but the water was gassed with room air (Po 2  18.4 kPa,  138 Torr). After the experiment, specimens were cooled on ice and killed by decapitation. Organs were removed, shock-frozen in liquid N 2 and stored at )80 °C until use. RNA extraction RNA samples from total goldfish or single organs were extracted using the RNeasy Mini Kit by Qiagen (Hilden, Germany). Tissues were weighed and homogenized in the required volume of RLT buffer (Qiagen). To avoid contami- nation with genomic DNA, a DNase digestion was per- formed on the Qiagen columns. Quality and amount of RNA were checked photometrically and with gel electrophoresis. cDNA amplification, cloning and sequencing Total RNA was extracted from goldfish brain, liver, heart, skeletal muscle, spleen, eyes and gills as described above. Partial or complete cDNA sequences of C. auratus Hba, Hbb, Mb1, Mb2, Ngb, Cygb1, acidic ribosomal protein (ARP; also known as rplp0), LDH-A and Cu ⁄ Zn-SOD- 1were amplified via RT-PCR with the OneStep RT-PCR kit (Qiagen) using degenerated or specific oligonucleotide primers (supplementary Table S1). The cDNA fragments were cloned into the pCR4-TOPO-TA (Invitrogen, Karlsruhe, Germany) or the pGEMTeasy vector (Promega, Mannheim, Germany). Poly(A) + RNA was purified from total RNA using the PolyATract TM kit (Promega); 5 lg poly(A) + RNA were used for the construction of a A. Roesner et al. Goldfish globins under hypoxia FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS 3639 directionally cloned cDNA expression library applying the Lambda ZAP-cDNA synthesis kit (Stratagene, Heidelberg, Germany) according to the manufacturer’s instruction. The library was then screened with digoxigenin-labeled cDNA fragments of the globins. Positive phage clones were con- verted to plasmid vectors using the material provided in the cDNA synthesis kit. cDNAs inserted in the pBK-CMV vector were sequenced on both strands by a commercial sequencing service (Genterprise, Mainz, Germany). In some cases, the incomplete clones were extended by 5¢- and 3¢-RACE (Invitrogen) with a series of nested oligonucleo- tide primers according to the manufacturer’s instructions. The sequences were obtained after the cloning of the PCR products into pCR4-TOPO-TA (Invitrogen) or pGEM-Teasy vector (Promega). Quantitative real-time RT-PCR RNA extractions and cDNA synthesis were carried out from tissues of single specimens. Quantitative real-time RT-PCR was performed according to a two-step protocol. First, total RNA was converted into cDNA employing Superscript II RNase H ) Reverse Transcriptase (Invitrogen) and an oligo(dT) 16 primer according to the manufacturer’s instructions. The cDNA samples were diluted with the same volume of DNase-free water. Real-time RT-PCR experi- ments were carried out on an ABI Prism 7000 SDS (Applied Biosystems, Darmstadt, Germany) using the Power SYBR Ò Green PCR Master Mix (Applied Biosystems). Levels of mRNA of ARP, LDH-A, SOD-1, Hba,Hbb, Mb1, Mb2, Ngb and Cygb1 were evaluated. To avoid amplification of genomic DNA, all primer pairs included one intron-span- ning oligonucleotide. The oligonucleotide primers were obtained from Sigma-Genosys (Hamburg, Germany) (sup- plementary Table S2). Reactions were run in triplicate with one or two repetitions, using 1 lL of diluted cDNA as tem- plate in a reaction volume of 25 lL. Primer concentrations were 0.13 lm for each oligonucleotide. The Taq DNA poly- merase was activated for 15 min at 95 ° C, followed by 40 cycles of a standard PCR protocol (15 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C). The efficiency of the reaction was measured by the slope of a standard curve. First evaluation of results was performed in the ABI Prism 7000 sds pro- gram; for normalization and calibration data were exported to qBase (http://www.medgen.ugent.be/qbase/). Final data analyses were carried out with the Microsoft Ò excel 2003 spreadsheet program (Microsoft, Redmond, WA, USA). The significance of the data was evaluated by Student’s t-test. Recombinant protein expression and antibody preparation The complete coding sequences of D. rerio Ngb was cloned into the pET3a and Mb into pET15b expression vectors (Novagen, Darmstadt, Germany) employing PCR-generated restriction sites. Plasmids were transformed into Escherichia coli BL21(DE3)pLys and grown at 25 °C in TBY medium (0.5% NaCl, 1% tryptone, 0.5% yeast extract, pH 7.4) containing 100 lgÆmL )1 ampicillin, 30 lgÆmL )1 chloram- phenicol and 1 mmolÆL )1 d-aminolevulinic acid. The culture was induced at D 600 = 0.8 by isopropyl-b-d-thiogalacto- pyranoside (0.4 mmolÆL )1 ). After 16 h, bacteria were har- vested by centrifugation and resuspended in 50 mmolÆL )1 Tris ⁄ HCl, pH 8.0, 1 mmolÆL )1 EDTA, 0.5 mmolÆ L )1 di- thiothreitol, 8 lgÆmL )1 DNase and 4 lgÆmL )1 RNase supplemented with CompleteÔ proteinase inhibitor mixture (Roche Applied Science, Mannheim, Germany) and Pefab- loc (Roth, Karlsruhe, Germany). The cells were broken by freeze–thaw cycles in fluid nitrogen followed by ultrasonica- tion. DNA and RNA were digested for 2 h at 37 °C. Cell debris was removed by centrifugation (1 h at 4 °Cat 10 000 g). Ngb was purified by ammonium sulfate precipi- tation, followed by DEAE ion-exchange column and size exclusion chromatography. His-tagged Mb was purified by affinity chromatography (Protino Ò Ni 2000 prepacked columns; Macherey and Nagel, Du ¨ ren, Germany). Final globin fractions were analyzed by gel electrophoresis, pooled, concentrated and stored frozen at )20 °C. Protein concentrations were determined using the Bradford [58] method. Purified recombinant D. rerio Ngb and Mb were used to raise a polyclonal antibody in rabbits. Specific Ngb antibodies were affinity-purified from crude rabbit serum using recombinant D. rerio Ngb coupled to a HiTrapÔ NHS-activated HP column (Amersham Biosciences, Munich, Germany) according to the manufacturer’s instruc- tions. The antibody was stored at )70 °C in 50 mmolÆL )1 Tris, 100 mmolÆL )1 glycine, pH 7.4 or supplemented with 0.1% NaN 3 at 4 °C. Protein extraction and western blotting Tissues were removed from the animal (goldfish or zebra- fish) and immediately homogenized in 1· NaCl ⁄ P i (140 mmolÆL )1 NaCl, 2.7 mmolÆL )1 KCl, 8.1 mmolÆL )1 Na 2 HPO 4 , 1.5 mmolÆL )1 KH 2 PO 4 ) by ultrasonication. The debris was precipitated by centrifugation for 10 min at 13 000 g at 4 °C and the supernatant was stored at ) 20 °C until use. Protein concentrations in the samples were deter- mined according to Bradford [58]. Protein extracts (100 lg) were diluted in sample buffer (31.25 mmolÆL )1 Tris ⁄ HCl, pH 6.8, 1% SDS, 2.5% b-mercaptoethanol, 5% glycerol) and heat-denatured for 5 min at 95 °C. Samples were applied to a 15% SDS-polyacrylamide gel and run at 100– 120 V. Proteins were transferred to a nitrocellulose mem- brane for 2 h at 0.8 mAÆcm )2 . Nonspecific binding sites were blocked by incubating for 45 min with 2% BSA in NaCl ⁄ Tris (10 mmolÆL )1 Tris, pH 7.4, 140 mmolÆ L )1 NaCl). Membranes were then incubated for 2 h with anti-Ngb or anti-zebrafish-Mb serum, both diluted 1 : 500 Goldfish globins under hypoxia A. Roesner et al. 3640 FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS in 2% BSA ⁄ NaCl ⁄ Tris, and washed four times for 5 min with NaCl ⁄ Tris. Membranes were incubated with the goat anti-(rabbit IgG) coupled with alkaline phosphatase (Dianova, Hamburg, Germany) for 1 h, diluted 1 : 10 000 in 2% BSA ⁄ NaCl ⁄ Tris, and washed as described above. Detection was carried out with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate salt as substrates. The membranes were scanned at 1200 dpi and the images were imported into the scion image program (version Beta 4.0.3). Protein levels were estimated by analy- ses of grey values. Mean gray values of the background of empty gel lanes were subtracted from the measurements of Ngb or Mb protein levels. Data were imported into Micro- soft Ò excel 2003 spreadsheet program (Microsoft). Statisti- cal analyses were performed by Student’s t-tests. Acknowledgements We thank F. Gerlach for his advice on the real-time PCR experiments and critical reading of the manu- script. This work has been supported by grants of the Deutsche Forschungsgemeinschaft (Bu956 ⁄ 5, Bu956 ⁄ 11 and Ha2103 ⁄ 3) and the Fonds der Chemischen Industrie. References 1 van den Thillart G & van Waarde A (1985) Teleosts in hypoxia: aspects of anaerobic metabolism. 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(1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding Anal Biochem 72, 248–254 Supplementary material The following supplementary material is available online: Goldfish globins under hypoxia Fig S1 Sequence of C auratus hemoglobin a cDNA (accession number AM933143) Fig S2 Sequence of C auratus hemoglobin b cDNA (accession... AM933144) Fig S3 Sequence of the C auratus myoglobin 1 cDNA (accession number AM747267) Fig S4 Sequence of C auratus myoglobin 2 cDNA (accession number AM747268) Fig S5 Partial sequence of C auratus neuroglobin cDNA (accession number AM933145) Fig S6 Partial sequence of C auratus cytoglobin 1 cDNA (acc no AM933146) Fig S7 Western blot quantification of neuroglobin and myoglobin under hypoxia Fig S8 Western... quantification of neuroglobin and myoglobin in goldfish and zebrafish Table S1 Oligonucleotide primer sequences used for cloning of goldfish globins Table S2 Oligonucleotide primer sequences used for quantitative real-time RT-PCR This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality... Mathews AJ, Moens L, Dewilde S & Brittain T (2006) The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c FEBS Lett 580, 4884– 4888 54 Wakasugi K, Nakano T & Morishima I (2003) Oxidized human neuroglobin acts as a heterotrimeric Galpha protein guanine nucleotide dissociation inhibitor J Biol Chem 278, 36505–36512 55 Hoppeler H & Vogt M (2001) Muscle tissue adaptations... (2001) Muscle tissue adaptations to hypoxia J Exp Biol 204, 3133–3139 56 Levine BD & Stray-Gundersen J (2001) The effects of altitude training are mediated primarily by acclimatization, rather than by hypoxic exercise Adv Exp Med Biol 502, 75–88 57 Sollid J, De Angelis P, Gundersen K & Nilsson GE (2003) Hypoxia induces adaptive and reversible gross morphological changes in crucian carp gills J Exp Biol... note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS 3643 . Mb and Ngb protein and mRNA levels increased significantly. The data suggest that these globins are involved in conveying hypoxia tolerance to zebrafish. Here we investigate the response of globin. [9,10]. Cyprinid fishes of the genus Carassius (the crucian carp Carassius carassius and its domestic Asian form, the goldfish C. auratus) routinely experience hypoxia and even anoxic phases in their. protein expression in goldfish as well as in zebrafish brain, suggesting that nonmuscle expression of Mb emerged before the diver- gence of D. rerio and Cyprininae within the lineage of the Cypriniformes.